Damage from reactive oxygen species (ROS) and perturbations in thiol redox cycling play a major role in aging, senescence, and apoptosis and drive the evolution of antioxidant defense (AOD) systems. However, AOD and redox maintenance incur metabolic and physiological costs, and a balance exists between the cost of damage and the cost of repair. Organisms that routinely experience highly stressful and mutagenic environments are under selection for an increased investment in developing novel means of AOD and redox maintenance. Bdelloid rotifers, small aquatic invertebrates of similar complexity to nematodes, are one such group. Bdelloids inhabit ephemerally aquatic environments and can survive repeated rounds of desiccation without a decrease in hydrated lifespan. In some species, females that have been through desiccation have increased lifespan and fecundity. Bdelloids are also among the most radiation resistant animals known, capable of repairing many hundreds of DNA double strand breaks after exposure to levels of ionizing radiation (IR) that would sterilize or kill any established animal model species. The extreme resistance of bdelloids to IR is almost certainly a consequence of their ability to tolerate the oxidative stress incurred during and recovering from desiccation. This is further supported by the observation that bdelloids are very resistant to hydrogen peroxide, with no observable effect at concentrations of H2O2 lethal to nematodes. Exposure to H2O2 at concentrations above the LC50 for nematodes increases lifespan and fecundity in bdelloids. Analysis of genomes and transcriptomes has revealed that bdelloids express multiple copies of genes for the synthesis and reduction of a glutathione-like polyamine dithiol, trypanothione, otherwise known only from kinetoplastid protozoans such as trypanosomes. This R21-scale project will characterize this novel thiol redox system and assess its contribution to different lifespan and fecundity outcomes after oxidative stress in rotifers and model organisms. The guiding hypothesis is that investigating a novel group of animals where redox maintenance AOD systems are maximized will provide new insights and approaches to increasing lifespan and healthspan. The first aim will test the hypothesis that trypanothione plays a major role in the bdelloid AOD and redox maintenance response to oxidative stress and aging, using a series of genetic and biochemical analyses. The second aim will test the hypothesis that the trypanothione system is sufficient to confer enhanced resistance to oxidative stress and is in part responsible for the observation of increased lifespan and fecundity, using RNAi to knock down trypanothione synthase in rotifers and by expressing the synthase and reductase in E. coli and C. elegans followed by exposure to oxidative stress. The long-term objective of this work is to establish bdelloid rotifers as a particularly advantageous model for comparative studies of redox maintenance and AOD systems as they relate to aging, with the goal of developing methods for enhancing these systems in other species, including standard aging models.